Engineered exosome for treating sepsis as well as construction method and application of engineered exosome

Abstract

The invention provides an engineered exosome for treating sepsis as well as a construction method and application of the engineered exosome, and belongs to the technical field of biological medicines. The invention provides an engineered exosome for treating sepsis. The engineered exosome comprises the following fusion proteins: a first fusion protein formed by a cryptochrome 2 protein and an exosome scaffold protein, a lysosome targeting peptide, and a second fusion protein formed by a Caspase 1 protein recruitment protein structural domain and a cryptochrome 2 protein ligand. The cell level and animal level prove that the engineered exosome can efficiently and accurately inhibit secretion of inflammatory factors, reduce inflammatory response and pathological conditions in a sepsis animal model, and restore animal functions of the sepsis model to a certain extent. Therefore, the engineered exosome provided by the invention provides a new means for treating sepsis, and has great market popularization and application values.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to an engineered exosome for treating sepsis, and a construction method and application thereof. Background Art

[0002] Sepsis is a systemic inflammatory response syndrome caused by infection. At the same time, burn sepsis is a common complication after deep burns with a high mortality rate. It has placed a heavy burden on patients and society. In recent decades, few innovative drugs have been launched for sepsis. Recombinant activated protein C, i.e., trombopag α, is the only drug approved by the FDA for sepsis in 2002, but it was withdrawn due to failure of post-marketing confirmation trials. At present, clinical treatment of sepsis is mainly based on the Sepsis-3.0 diagnostic criteria, with early fluid resuscitation, use of anti-infective drugs, neuromuscular blockers, and organ support. However, after strict treatment in accordance with the standard regimen, the mortality rate of patients with sepsis has not been significantly reduced, and there is a high unmet clinical need. Therefore, there is currently no effective treatment for sepsis. The treatment of sepsis is an important clinical problem facing medicine, and understanding its pathogenesis and individualized treatment are crucial to the treatment of sepsis.

[0003] Excessive release of proinflammatory factors caused by macrophage activation is the core pathological mechanism of sepsis. Caspase 1 protein is an important regulator of proinflammatory factor release. Caspase 1 regulates the inflammatory response of cells by promoting the processing and release of inflammatory factors. Caspase 1 activated by inflammasomes can cleave IL-1β precursor (pro-IL-1β) and IL-18 precursor (pro-IL-18) to form active IL-1β and IL-18. Caspase 1 also promotes the release of these processed and mature inflammatory factors. IL-1β and IL-18 belong to the IL-1 superfamily and are important proinflammatory factors in cells. Both can activate the downstream NF-κB signaling pathway by binding to their respective receptors, thereby promoting the release of some inflammatory factors, such as IL-3, IL-5, IL-6 and IL-13. Previous studies mainly degraded Caspase 1 at the RNA level, which has many disadvantages. First, systemic administration cannot accurately target macrophages. At the same time, macrophages are rich in lysosomes. After RNA molecules reach macrophages, they are quickly phagocytosed by macrophage lysosomes and cannot play a role, resulting in low delivery efficiency. Secondly, RNA intervention is prone to off-target effects and affect normal cell functions, so it takes a long time to take effect, resulting in low treatment efficiency. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide an engineered exosome for the treatment of sepsis, which is based on optogenetics and simultaneously loaded with functional elements that target macrophage lysosomes and recruit Caspase 1, to achieve the degradation of Caspase 1 protein in macrophages, thereby regulating the inflammatory response in patients with sepsis.

[0005] The present invention provides an engineered exosome for treating sepsis, comprising the following fusion proteins: a first fusion protein formed by a cryptochrome 2 protein and an exosome scaffold protein and a second fusion protein formed by a lysosome targeting peptide, a Caspase 1 protein recruiting protein domain and a cryptochrome 2 protein ligand.

[0006] Preferably, the amino acid sequence of the lysosome targeting peptide is as shown in SEQ ID NO: 1;

[0007] The amino acid sequence of the Caspase 1 protein recruitment protein domain is shown in SEQ ID NO: 2;

[0008] The amino acid sequence of the cryptochrome 2 protein ligand is shown in SEQ ID NO:3.

[0009] Preferably, the amino acid sequence of the second fusion protein is as shown in SEQ ID NO:4.

[0010] Preferably, the amino acid sequence of the cryptochrome 2 protein is as shown in SEQ ID NO: 5;

[0011] The amino acid sequence of the exosome scaffold protein is shown in SEQ ID NO:6.

[0012] Preferably, the amino acid sequence of the first fusion protein is as shown in SEQ ID NO:7.

[0013] The present invention provides a method for constructing engineered exosomes for treating sepsis, comprising the following steps:

[0014] Constructing a first recombinant plasmid containing a first fusion gene formed by a cryptochrome 2 gene and an exosome scaffold protein gene;

[0015] constructing a second recombinant plasmid containing a second fusion gene formed by a lysosomal targeting peptide sequence, a Caspase 1 protein recruitment protein domain sequence and a cryptochrome 2 protein ligand gene;

[0016] The first recombinant plasmid and the second recombinant plasmid were transfected into HEK293T cells, and after blue light stimulation, exosomes were extracted to obtain engineered exosomes.

[0017] Preferably, the nucleotide sequence of the first fusion gene is shown in SEQ ID NO: 8;

[0018] The nucleotide sequence of the second fusion gene is shown in SEQ ID NO:9.

[0019] Preferably, the method for extracting exosomes is to extract by gradient ultracentrifugation;

[0020] The program of the gradient ultracentrifugation is: 2000×g, 30 min; 10000×g, 70 min; 100000×g, 120 min.

[0021] The present invention provides the use of the engineered exosomes for treating sepsis or the engineered exosomes obtained by the construction method in preparing a drug for preventing and / or treating sepsis.

[0022] The present invention provides a drug for preventing and / or treating sepsis, comprising the engineered exosomes for treating sepsis or the engineered exosomes obtained by the construction method and medically acceptable excipients.

[0023] The present invention provides an engineered exosome for treating sepsis, comprising the following fusion proteins: a first fusion protein formed by a cryptochrome 2 protein and an exosome scaffold protein and a second fusion protein formed by a lysosome targeting peptide, a Caspase 1 protein recruiting protein domain and a cryptochrome 2 protein ligand. Aiming at the characteristic that most of the engineered exosomes will be phagocytosed by macrophages after entering the body, the present invention uses exosomes as carriers and genetically engineers the contents of exosomes. First, the exosome scaffold protein is fused with the cryptochrome 2 protein to achieve direct encapsulation of the cryptochrome 2 protein in the exosomes. At the same time, the lysosome targeting peptide, the Caspase 1 protein recruitment protein domain and the cryptochrome 2 protein ligand are fused to form a second fusion protein, wherein the cryptochrome 2 protein ligand binds to the cryptochrome 2 protein within a sub-second time under the stimulation of blue light, thereby ensuring that the second fusion protein is accurately encapsulated in the exosomes. Under the action of the lysosome targeting peptide, the exosomes entering the macrophages can efficiently target the lysosomes. At the same time, the Caspase 1 protein recruitment protein domain can recruit the Caspase 1 protein in the macrophages, thereby utilizing the dissolution characteristics of the lysosomes to degrade the recruited Caspase 1 protein. Based on the fact that the Caspase 1 protein is a key protein that promotes the processing and secretion of inflammatory factors by macrophages, the degradation of the Caspase 1 protein effectively inhibits the inflammatory response in the macrophages and reduces the processing and release of inflammatory factors. The results of the examples of the present invention show that the engineered exosomes can efficiently and accurately inhibit the secretion of inflammatory factors, reduce the inflammatory response and alleviate the pathological conditions in the animal model of sepsis, and restore the lung function of the sepsis model animal to a certain extent. It can be seen that the engineered exosomes provided by the present invention provide a new means for the treatment of sepsis and have great market promotion and application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Schematic diagram of the structure of the first fusion gene and the second fusion gene constructed in the present invention;

[0025] Figure 2 A diagram of the working principle of the engineered exosomes constructed for the present invention;

[0026] Figure 3 A schematic diagram of the construction process of the engineered exosomes provided by the present invention;

[0027] Figure 4 The characterization results of the engineered exosomes provided by the present invention, wherein A is the morphology of Exo-LD observed by transmission electron microscopy, B is the result of the number and size of Exo-LD particles detected by nanoparticle tracking analysis, and C is the result of the identification of exosome markers by Western Blot experiment;

[0028] Figure 5Identification of the loading capacity of engineered exosomes (A) and marker identification results (B);

[0029] Figure 6 The immunoprecipitation results of Caspase 1 protein and GCE-CTM protein (A) and the delivery results of Caspase 1 detected by laser confocal microscopy (B);

[0030] Figure 7 The results are for the degradation of Caspase 1 in macrophages under simulated sepsis conditions;

[0031] Figure 8 ELISA experiments were performed to detect the secretion of IL18 (A) and IL1β in the cell supernatant (B), and the expression of Il18 (C) and Il1β (D) after Exo-LD treatment;

[0032] Fig. 9 The expression of Caspase 1, IL18 and IL1β proteins in different organs; A is lung, B is kidney, C is liver, D is spleen, and E is heart;

[0033] Fig.10 Results of ELISA test to detect the secretion of IL18 protein (A) and IL1β protein (B) in serum;

[0034] Fig.11 The results of qPCR experiments to detect the expression of Il18 and Il1β in different organs and tissues of septic mice in each treatment group; A is the heart, B is the liver, C is the spleen, D is the lung, and E is the kidney;

[0035] Fig.12 The inflammation and pathological results of various organs in septic mice; A is the pathological section result, B is the ALT test result, C is the Cr test result, and D is the lung tissue weight ratio result;

[0036] Fig.13 Figure 2 shows the survival curves of septic mice in different treatment groups. DETAILED DESCRIPTION

[0037] The present invention provides an engineered exosome for treating sepsis, comprising the following fusion proteins: a first fusion protein formed by cryptochrome 2 protein and exosome scaffold protein and a second fusion protein formed by lysosomal targeting peptide, Caspase 1 protein recruitment protein domain and cryptochrome 2 protein ligand, as shown in the schematic diagram Figure 1 The schematic diagram of the working principle of the engineered exosomes is shown in Figure 2 .

[0038] In the present invention, in the second fusion protein, the amino acid sequence of the lysosomal targeting peptide is preferably as shown in SEQ ID NO: 1 (KFERQKILDQRFFE). The amino acid sequence of the Caspase 1 protein recruitment protein domain is preferably as shown in SEQ ID NO: 2 (AADFQGLYAEVKACSSELESLEMELRQQILVNIGKILQDQPSMEALEASLGQGLCSGGQV EPLDGPAGCILECLVLDSGELVPELAAPIFYLLGALAVLSETQQQLLAKALETTVLSKQLELVKHV LEQSTPWQEQSSVSLPTVLLGDCWDEKNPTWVLLEECGLRLQVESPQVHWEPTSLIPTSALYASL FLLSSLGQKPC); the amino acid sequence of the cryptochrome 2 protein ligand is preferably as shown in SEQ ID NO: 3 (NGAIG GDLLLNFPDMSVLERQRAHLKYLNPTFDSPLAGFFADSSMITGGEMDSYLSTAGLNLPMMYGET TVEGDSRLSISPETTLGTGNFKKRKFDTETKDCNEKKKKMTMNRDDLVEEGEEEKSKITEQNNGS In the second fusion protein, it is preferred that a Flag tag is also included to facilitate subsequent detection.The amino acid sequence of the second fusion protein is preferably as shown in SEQ ID NO: 4 (NGAIGGDLLLNFPDMSVLERQRAHLKYLNPTFDSPLAGFFADSSMITGGEMDSYLSTAGLNLPMMYGETTVEGDSRLSISPETTLGTGNFKKRKFDTETKDCNEKKKKMTMNRDDLVEEGEEEKSKITEQNNGSTKSIKKMKHKAKKEENNFSNDSSKVTKELEKTDYIDYKDDDDKDYKDDDDKDYKDDDDKGGGGSGGGGSGGGGSAADFQGLYAEVKACSSELESL EMELRQQILVNIGKILQDQPSMEALEASLGQGLCSGGQVEPLDGPAGCILECLVLDSGELVPELAAPIFYLLGALAVLSETQQQLLAKALETTVLSKQLELVKHVLEQSTPWQEQSSVSLPTVLLGDCWDEKNPTWVLLEECGLRLQVESPQVHWEPTSLIPTSALYASLFLLSSLGQKPCGGGGSGGGGSGGGSKFERQKILDQRFFE). The Caspase 1 protein recruiting protein domain can recruit Caspase 1 protein, and the protein domain GCE that binds to Caspase 1. The lysosomal targeting peptide is CTM, which is a lysosomal targeting peptide with a molecular weight of 1.88 kDa. Its function is to efficiently and specifically target the lysosomes in macrophages so that the recruited Caspase 1 protein can be degraded by the lysosomes. The cryptochrome 2 protein ligand can bind to the cryptochrome 2 protein within sub-seconds upon stimulation by blue light, and depolymerizes with the cryptochrome 2 protein under dark conditions, and acts by reversibly binding to the cryptochrome 2 protein.

[0039] In the present invention, the amino acid sequence of the cryptochrome 2 protein is preferably as shown in SEQ ID NO: 5 (KMDKKTIVWFRRDLRI). The amino acid sequence of the exosome scaffold protein is preferably as shown in SEQ ID NO: 6 (GPIFNASVHSDTPSVIRGDLIKLFCIITVEGAALDPDDMAFDVSWFAVHSFGLDKAPVLLSSLDRKGIVTTSRRDWKSDLS LERVSVLEFLLQVHGSEDQDFGNYYCSVTPWVKSPTGSWQKEAEIHSKPVFITVKMDVLNAFKY PLLIGVGLSTVIGLLSCLIGYCSSHWCCKKEVQETRRERRRLMSMEMD).The exosome scaffold protein in the first fusion protein is specifically expressed on the exosome membrane. When the exosome scaffold protein is fused with the cryptochrome 2 protein and expressed, the cryptochrome 2 protein is ensured to enter the exosome, and the cryptochrome 2 protein is used to reversibly bind to the cryptochrome 2 protein ligand, thereby achieving efficient expression of the lysosomal targeting peptide and the Caspase 1 protein recruitment protein domain in the exosome. The amino acid sequence of the first fusion protein is preferably as shown in SEQ ID NO: 7 (GPIFNASVHSDTPSVIRGDLIKLFCIITVEGAALDPDDMAFDV).

[0040] The present invention provides a method for constructing engineered exosomes for treating sepsis, comprising the following steps:

[0041] Constructing a first recombinant plasmid containing a first fusion gene formed by a cryptochrome 2 gene and an exosome scaffold protein gene;

[0042] constructing a second recombinant plasmid containing a second fusion gene formed by a lysosomal targeting peptide sequence, a Caspase 1 protein recruitment protein domain sequence and a cryptochrome 2 protein ligand gene;

[0043] The first recombinant plasmid and the second recombinant plasmid are transfected into a mammal, and after blue light stimulation, exosomes are extracted to obtain engineered exosomes.

[0044]

[0045]

[0046] The present invention has no special restrictions on the method of transfecting HEK293T cells, and a transfection method well known in the art can be used, such as transient transfection. After the transfection, the transfected HEK293T cells are preferably cultured. The culture method preferably replaces the serum-free culture medium 6 hours after transfection. The wavelength of the blue light is preferably 460nm. The treatment time of the blue light is preferably 46 to 50 hours, more preferably 48 hours. Exosomes are extracted 45 to 50 hours after the blue light treatment. The method for extracting exosomes is preferably extracted by gradient ultracentrifugation. The program of the gradient ultracentrifugation is: 2000×g, 30min; 10000×g, 70min; 100000×g, 120min.

[0047] In one embodiment of the present invention, transmission electron microscopy was used to observe the prepared engineered exosomes, and nanoparticle tracking analysis detected that the particle size of the engineered exosomes ranged from 40 to 160 nm, with an average particle size of 120 nm and a spherical shape. At the same time, Western Blot was used to identify the markers of the engineered exosomes, and it was found that the expression

[0048] The present invention provides the use of the engineered exosomes for treating sepsis or the engineered exosomes obtained by the construction method in preparing a drug for preventing and / or treating sepsis.

[0049] In one embodiment of the present invention, LPS-stimulated macrophages were co-incubated with the engineered exosomes, and the results showed that the Caspase 1 protein content in the treatment group was reduced, and the IL18 and IL1β protein content was reduced. At the same time, the present invention also used a severe sepsis animal model as an object to evaluate the efficacy of the engineered exosomes. The results showed that after treatment with engineered exosomes, the heart of septic mice recovered, the lung function recovered, and the HE staining results of the heart, liver, spleen, lung, kidney and intestine showed that the pathological symptoms of each organ were alleviated. The statistical survival rate results showed that the treatment of the engineered exosomes was beneficial to reduce the mortality rate.

[0050] The present invention provides a drug for preventing and / or treating sepsis, comprising the engineered exosomes for treating sepsis or the engineered exosomes obtained by the construction method and medically acceptable excipients.

[0051] The following is a detailed description of an engineered exosome for treating sepsis provided by the present invention, as well as a construction method and application thereof, in conjunction with the embodiments. However, they should not be construed as limiting the scope of protection of the present invention.

[0052] The detection primers involved in the present invention are shown in Table 1.

[0053] Table 1 Primer sequence information

[0054]

[0055] Example 1

[0056] A method for preparing engineered exosomes for treating sepsis

[0057] 1. Construction of recombinant plasmid

[0058] The cryptochrome 2 gene and the exosome scaffold protein gene were fused to form a first fusion gene (SEQ ID NO: 8), and the first fusion gene was cloned into the XbaI / Kpn1 multiple cloning site in the pcDNA3.1(-) plasmid to form a first recombinant plasmid; the restriction site XbaI was added upstream of the PTGFRN-CRY2 fragment, and the restriction site Kpn1 sequence was added after the stop codon.

[0059] The lysosomal targeting peptide sequence, the Caspase 1 protein recruitment protein domain sequence, the cryptochrome 2 protein ligand gene and the tag sequence are fused to form a second fusion gene (SEQ ID NO: 9), and the second fusion gene is cloned into the pcDNA3.1(-) plasmid to form a second recombinant plasmid;

[0060] The first recombinant plasmid and the second recombinant plasmid are respectively transfected into competent Escherichia coli, and positive transformants are obtained through screening, culture, colony identification and sequencing;

[0061] The first recombinant plasmid and the second recombinant plasmid are extracted from the positive transformants respectively;

[0062] The first recombinant plasmid and the second recombinant plasmid were transiently inoculated into HEK293T cells, and the serum-free medium was replaced after 6 hours, and a blue light treatment with a wavelength of 460nm was applied for 48 hours. After 48 hours of blue light treatment, ultracentrifugation (300×g for 10 minutes; 2000×g for 30 minutes; 10000×g for 70 minutes; 100000×g for 120 minutes) was performed to obtain engineered exosomes (Exo-LD), see Figure 3 .

[0063] Example 2

[0064] Characterization of engineered exosomes

[0065] The engineered exosomes prepared in Example 1 were observed by transmission electron microscopy, and Exo-Ctrl was used as a control. The results showed that the morphology was round (A), and the nanoparticle tracking analysis detected that the particle size of Exo-LD was 40-160 nm, with an average particle size of 100 nm (B).

[0066] Western Blot assay was used to detect markers of engineered exosomes. The specific method is as follows:

[0067] 15 μg of engineered exosomes were loaded onto 12% SDS-PAGE gel electrophoresis and treated at 120 V constant voltage for 1 h 40 min;

[0068] Transfer (NC membrane) 200 mA, 2 h 30 min, ice bath;

[0069] 8% skim milk blocking for 2 h at room temperature;

[0070] Primary antibody incubation at 4°C overnight;

[0071] Incubate with secondary antibody at room temperature for 1 h

[0072] Luminescence detection.

[0073] Results Figure 4 Middle C. The results showed that the morphology and size distribution of engineered exosomes did not change significantly compared with the control exosomes (Exo-Ctrl, Exo-Turn off).

[0074] Example 3

[0075] Verification of loading capacity, marker detection and transfection ability of engineered exosomes

[0076] 1. Loading capability verification

[0077] The Western Blot method was used to detect the loading capacity of exosomes. The specific method is as follows:

[0078] 15 μg of engineered exosomes and control exosomes (Exo-Turn off) were loaded onto 12% SDS-PAGE gel electrophoresis and treated at a constant voltage of 120 V for 1 h 40 min;

[0079] Transfer (NC membrane): 200 mA current flow for 2 h 30 min, ice bath;

[0080] 8% skim milk blocking for 2 h at room temperature;

[0081] Primary antibody incubation at 4°C overnight;

[0082] Incubate with secondary antibody at room temperature for 1 h

[0083] Luminescence detection.

[0084] Results Figure 5 Middle A. The results showed that compared with the control exosomes (Exo-Turn off), the FLAG protein was highly abundantly expressed in the engineered exosomes. This indicates that the use of EXPLORs technology in the generation of engineered exosomes improves the loading efficiency of engineered exosomes.

[0085] 2. Marker Detection

[0086] Exosome markers were identified by Western Blot assay. Figure 5 In Figure B, the results showed that when the sample loading amount was the same, FLAG protein and CRY2 protein were not expressed in the cells and exosomes of the ctrl group, while in the other two groups.

[0087] Example 4

[0088] 1. Use Co-IP to verify the binding ability of CIBN-GCE-CTM (second fusion protein) with Caspase 1

[0089] The Co-IP kit produced by Bio-Tech was used to detect the exosome binding ability. For specific operation methods, please refer to the instructions.

[0090] Results Figure 6 Middle A. The results showed that from the immunoprecipitation results, Caspase 1 and CIBN-3XFLAG-GCE-3XFLAG-CTM fusion protein had obvious protein bands, proving that Caspase 1 protein interacted with GCE-CTM protein.

[0091] 2. Laser confocal microscopy to observe the lysosomal targeting effect of CIBN-GCE-CTM

[0092] To further observe whether exosomes can successfully target Caspase 1 to lysosomes, Caspase 1 labeled with an immunofluorescent secondary antibody (green) was visualized. RAW264.7 cells were plated on confocal culture dishes two days in advance and stimulated with LPS on the second day, and incubated with exosomes. After 24 hours, they were incubated with Caspase 1 primary antibody (Wuhan Tri-Tac, 1; 50) overnight and then washed three times with PBS. The secondary antibody (Wuhan Tri-Tac, 1; 200) was used for 2 hours. At the end of the incubation, the cells were rinsed three times with PBS; lysosomes were stained with lysotracker (Invitrogen, USA 1: 10000) for 30 minutes, and the cells were rinsed three times with PBS. The cell nuclei were stained with Hoechst (Invitrogen, USA). At the end of the incubation, the cells were rinsed with PBS and imaged under a confocal microscope.

[0093] The results are as follows Figure 6 Center B shows that Caspase 1 can be successfully delivered to macrophage lysosomes.

[0094] Example 5

[0095] Verification of the function of engineered exosomes at the cellular level

[0096] 1. Detection of Caspase 1 degradation and IL18 and IL1β protein expression after engineered exosome treatment at the cellular level

[0097] The macrophage proteins were extracted after LPS stimulation and different treatments were applied. The degradation of Caspase 1 after engineered exosome treatment was detected by Western blot, and the expression of IL18 and IL1β proteins was detected.

[0098] Results Figure 7 The results showed that Caspase 1, IL18, and IL1β proteins were highly expressed in the LPS-treated group and the Exo-Ctrl-treated group compared with the engineered exosome-treated group, and there was no significant difference in expression between the two groups. This suggests that engineered exosome treatment can effectively inhibit the expression of Caspase1, IL18, and IL1β proteins in macrophages after LPS stimulation.

[0099] 2. Detection of IL18 and IL1β secretion in cell supernatant by ELISA

[0100] The IL18 Elisa kit (purchased from Lianke Bio, catalog number: EK218, Mouse IL-18 ELISA Kit detection kit (enzyme-linked immunosorbent assay)) was used to detect the IL18 secretion in the supernatant of cells incubated with engineered exosomes.

[0101] The IL1β ELISA kit (purchased from Lianke Bio, catalog number: EK201B, Mouse IL-1β ELISA Kit detection kit (enzyme-linked immunosorbent assay)) was used to detect the secretion of IL1β in the supernatant of cells incubated with engineered exosomes.

[0102] Results Figure 8 A and B. The results showed that compared with the control exosomes (Exo-Ctrl), the engineered exosomes treatment significantly reduced the concentrations of IL18 and IL1β in the cell supernatant.

[0103] 3. Detection of Il18 and Il1β expression levels by qPCR

[0104] Total RNA was extracted using TRIzol (Invitrogen, USA). According to the manufacturer's instructions, the target RNA was reverse transcribed using the Transcriptor First-strand cDNA Synthesis Kit. The reverse transcription system was 1 μg of total RNA, 4 μl of 5× PrimeScript RT Master Mix, and ddH2O to 20 μl. The reverse transcription conditions were 42°C for 15 min; 85°C for 5 min; and 4°C for insulation. FastStart Essential DNA Green Master (Indianapolis, IN, USA) was used for 20 μL of qPCR reaction. Relative gene expression was normalized to GAPDH, and the -ΔCt method was used for quantitative comparison.

[0105] Results Figure 8 As shown in C and D. The results showed that the expression of Il18 and Il1β was significantly decreased after Exo-LD treatment.

[0106] Example 6

[0107] Functional verification of Exo-LD in animal experiments

[0108] 1. Preparation of DiR-labeled Exo-LD

[0109] DIR was added to Exo-LD at a volume ratio of 1:500, incubated at 37°C for 5 min in the dark; then incubated at 4°C for 15 min in the dark; then centrifuged at 4°C and 12000 g for 10 min in the dark; discarded the supernatant, and resuspended the precipitate with 1× PBS to obtain DiR-labeled Exo-LD.

[0110] 2. Animal efficacy evaluation experiment

[0111] Cecal ligation and puncture (CLP) was performed on C57BL / 6 mice to establish an in vivo mouse sepsis model (reference: Dejager, L., Pinheiro, I., Dejonckheere, E., & Libert, C. (2011). Cecal ligation and puncture: the gold standard model for polymicrobial sepsis?. Trends in microbiology, 19 (4), 198-208.). The animal model was successfully established when the animal developed fever, significantly increased heart rate and respiratory rate, increased oral and nasal secretions, lethargy, huddling, erect hair, less movement, refusal to eat or less food, and secretions appeared in the corners of the eyes. The model group was injected with DiR-labeled Exo-LD through the tail vein.

[0112] Example 7

[0113] Detection of Caspase 1 degradation and IL18 and IL1β protein expression after engineered exosome treatment at the animal level

[0114] Proteins from various tissues were extracted, and Western blot was used to detect the degradation of Caspase 1 in various organs (heart, liver, spleen, lung, and kidney) of Exo-LD, and the expression of IL18 and IL1β proteins was detected.

[0115] Results Fig. 9 The results showed that Caspase 1, IL18, and IL1β proteins were highly expressed in the heart, liver, spleen, kidney, and lung of mice in the PBS-treated group and the Exo-Ctrl-treated group compared with the engineered exosome-treated group, and there was no significant difference in expression between the two groups. This suggests that engineered exosome treatment can effectively inhibit the expression of Caspase 1, IL18, and IL1β proteins in the organs (heart, liver, spleen, kidney, and lung) of CLP mice.

[0116] Example 8

[0117] The above ELISA experiment was used to detect the secretion of IL18 and IL1β proteins in serum. IL18 and IL1β are two downstream proteins induced by Caspase 1. The detection of their concentrations can reflect the degree of activation of Caspase 1 for characterization.

[0118] Results Fig.10 The results showed that the engineered exosome treatment significantly alleviated the concentrations of IL18 and IL1β in the serum of CLP mice compared with the control exosomes (Exo-Ctrl).

[0119] Example 9

[0120] Detection of the effect of engineered exosomes on the expression level of pro-inflammatory factors after treatment at the animal level

[0121] Total RNA was extracted using TRIzol (Invitrogen, USA). The target RNA was reverse transcribed using the Transcriptor First-strand cDNA Synthesis Kit according to the manufacturer's instructions. FastStart Essential DNA Green Master (Indianapolis, IN, USA) was used for 20 μL qPCR reaction. Relative gene expression was normalized to GAPDH and quantitatively compared using the -ΔCt method.

[0122] The results showed that the expression levels of Il18 and Il1β in central liver, spleen, lung and kidney tissues were significantly decreased after Exo-LD treatment.

[0123] RNA was extracted from each tissue and the expression levels of pro-inflammatory factors Il18 and Il1β were detected by qPCR (see Fig.11 (Medium AE).

[0124] Example 10

[0125] The mortality rate of mice within 48 hours was observed by CLP and tail vein injection of Exo-LD, and the heart, liver, spleen, lung, kidney and other tissues were collected for hematoxylin-eosin staining to observe the degree of organ damage (A). The lung wet / dry ratio, creatine (Cr) and alanine aminotransferase (ALT) of CLP mice after Exo-LD treatment were detected (BD).

[0126] A severe sepsis model was established by cecal ligation and puncture (CLP). Before all operations, mice were anesthetized by intraperitoneal injection of sodium pentobarbital (1%). After anesthesia, the peritoneum was opened aseptically, 75% of the cecum was ligated with a 4.0 silk suture end, and punctured once with a 21-gauge needle. A small amount of feces was squeezed out from the perforation site to ensure the patency of the perforation. The cecum was returned to the abdominal cavity and the abdominal incision was sutured with 4.0 silk. After CLP, fluid resuscitation was performed immediately with preheated saline (50 ml / kg subcutaneously). Except for the cecal ligation and puncture, the other steps of the sham group were the same.

[0127] C57 / BL6N mice (male, 6-8 weeks) were randomly divided into 4 groups for tail vein injection, namely Sham group, CLP+PBS group, CLP+Exo-Ctrl group, and CLP+Exo-LD group.

[0128] The sham group received no treatment.

[0129] CLP+PBS group: After the mice underwent cecal ligation and puncture (CLP) to simulate sepsis, PBS was injected into the mice at a dose of 4 μg / g once every 6 hours for a total of 4 injections after obvious symptoms appeared.

[0130] The CLP+Exo-Ctrl group consisted of mice that underwent cecal ligation and puncture (CLP) to simulate sepsis. After obvious symptoms appeared, Exo-Ctrl was injected into the mice at a dose of 4 μg / g, once every 6 hours, for a total of 4 injections.

[0131] CLP+Exo - LD group: After the mice underwent cecal ligation and puncture (CLP) to simulate sepsis, after obvious symptoms appeared, Exo-LD was injected into the mice at a dose of 4 μg / g, once every 6 hours, for a total of 4 injections.

[0132] Afterwards, blood biochemistry tests (ALT, Cr) were performed on the serum of each group of mice to evaluate the liver and kidney functions under different treatments.

[0133] The results showed that after Exo-LD treatment, the liver and kidney functions of septic mice were restored.

[0134] Afterwards, the lungs of each group of mice were taken out and weighed, which was recorded as wet weight; then they were dried in an oven for 72 hours and weighed, which was recorded as dry weight; the ratio of lung wet weight to dry weight was calculated, and the lung function under different treatments was evaluated.

[0135] The results showed that the lung function of septic mice was restored after treatment with Exo-LD.

[0136] Then, the organs (heart, liver, spleen, lung, kidney and intestine) of each group of mice were stained with HE to observe the pathological changes of each organ under different treatments.

[0137] The results showed that after Exo-LD treatment, the inflammation and pathological changes of various organs in septic mice were significantly alleviated compared with those in the CLP+PBS group and CLP+Exo-Ctrl group (see Fig.12 ).

[0138] Example 12

[0139] The mortality rate of mice was observed within 48 hours by CLP and tail vein injection of Exo-LD

[0140] A severe sepsis model was established by cecal ligation and puncture (CLP). Before all operations, mice were anesthetized by intraperitoneal injection of sodium pentobarbital (1%). After anesthesia, the peritoneum was opened aseptically, 75% of the cecum was ligated with a 4.0 silk suture end, and punctured once with a 21-gauge needle. A small amount of feces was squeezed out from the perforation site to ensure the patency of the perforation. The cecum was returned to the abdominal cavity and the abdominal incision was sutured with 4.0 silk. After CLP, fluid resuscitation was performed immediately with preheated saline (50 ml / kg subcutaneously). Except for the cecal ligation and puncture, the other steps of the sham group were the same.

[0141] C57 / BL6N mice (male, 6-8 weeks) were randomly divided into 4 groups for tail vein injection, namely Sham group, CLP+PBS group, CLP+Exo-Ctrl group, and CLP+Exo-LD group.

[0142] The sham group received no treatment.

[0143] CLP+PBS group: After the mice underwent cecal ligation and puncture (CLP) to simulate sepsis, PBS was injected into the mice at a dose of 4 μg / g once every 6 hours for a total of 4 injections after obvious symptoms appeared.

[0144] The CLP+Exo-Ctrl group consisted of mice that underwent cecal ligation and puncture (CLP) to simulate sepsis. After obvious symptoms appeared, Exo-Ctrl was injected into the mice at a dose of 4 μg / g, once every 6 hours, for a total of 4 injections.

[0145] CLP+Exo - LD group: After the mice underwent cecal ligation and puncture (CLP) to simulate sepsis, after obvious symptoms appeared, Exo-LD was injected into the mice at a dose of 4 μg / g, once every 6 hours, for a total of 4 injections.

[0146] Results Fig.13 The mortality rate of mice was effectively reduced after treatment with engineered exosomes.

[0147] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. An engineered exosome for treating sepsis, characterized in that: The invention comprises the following fusion proteins: a first fusion protein formed by cryptochrome 2 protein and exosome scaffold protein and a second fusion protein formed by lysosome targeting peptide, Caspase 1 protein recruiting protein domain and cryptochrome 2 protein ligand.

2. The engineered exosomes for treating sepsis according to claim 1, characterized in that: The amino acid sequence of the lysosome targeting peptide is shown in SEQ ID NO: 1; The amino acid sequence of the Caspase 1 protein recruitment protein domain is shown in SEQ ID NO: 2; The amino acid sequence of the cryptochrome 2 protein ligand is shown in SEQ ID NO:

3.

3. The engineered exosomes for treating sepsis according to claim 1 or 2, characterized in that: The amino acid sequence of the second fusion protein is shown in SEQ ID NO:

4.

4. The engineered exosomes for treating sepsis according to claim 1, characterized in that: The amino acid sequence of the cryptochrome 2 protein is shown in SEQ ID NO: 5; The amino acid sequence of the exosome scaffold protein is shown in SEQ ID NO:

6.

5. The engineered exosomes for treating sepsis according to claim 1 or 4, characterized in that: The amino acid sequence of the first fusion protein is shown in SEQ ID NO:

7.

6. A method for constructing the engineered exosomes according to any one of claims 1 to 5, characterized in that: The following steps are involved: Constructing a first recombinant plasmid containing a first fusion gene formed by a cryptochrome 2 gene and an exosome scaffold protein gene; constructing a second recombinant plasmid containing a second fusion gene formed by a lysosomal targeting peptide sequence, a Caspase 1 protein recruitment protein domain sequence and a cryptochrome 2 protein ligand gene; The first recombinant plasmid and the second recombinant plasmid are transfected into recipient cells, and after blue light stimulation, exosomes are extracted to obtain engineered exosomes.

7. The construction method according to claim 6, characterized in that: The nucleotide sequence of the first fusion gene is shown in SEQ ID NO: 8; The nucleotide sequence of the second fusion gene is shown in SEQ ID NO:

9.

8. The construction method according to claim 6, characterized in that: The method for extracting exosomes is to extract by gradient ultracentrifugation; The program of the gradient ultracentrifugation is: 2000×g, 30 min; 10000×g, 70 min; 100000×g, 120 min.

9. Use of the engineered exosomes for treating sepsis according to any one of claims 1 to 5 or the engineered exosomes obtained by the construction method according to any one of claims 6 to 8 in the preparation of a drug for preventing and / or treating sepsis.

10. A drug for preventing and / or treating sepsis, characterized in that: The invention comprises the engineered exosomes for treating sepsis as described in any one of claims 1 to 5 or the engineered exosomes obtained by the construction method as described in any one of claims 6 to 8 and medically acceptable excipients.

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